The present invention relates to an immunological detection method capable of performing detection of an analyte in a test sample and a kit used therefor. More particularly, the present invention relates to immunochromatography comprising the step of amplifying a detection signal.
In an immunological analysis of a biological sample, or the like, a method for carrying out its detection quickly and simply includes immunochromatography. This method generally comprises the steps as described below. Concretely, when a mixture comprising a liquid of a test sample and a labeled antibody capable of specifically binding to an analyte is absorbed and developed from one end of a test strip comprising a water-absorbent substrate having a capture region immobilized with an antibody capable of specifically binding to the analyte, a labeled antibody-analyte complex formed in the mixture is bound to the immobilized antibody, thereby capturing the complex on the capture region. Therefore, the analyte in the liquid of a test sample can be determined by assaying the labeled antibody bound to the capture region.
In addition, as a method for obtaining a detection signal by the immunochromatography mentioned above with a higher sensitivity, a method using two kinds of labeled antibodies is disclosed in Japanese Patent Laid-Open No. Hei 10-062419. In other words, the construction is such that a first labeled antibody and a second labeled antibody are respectively positioned (absorbed) as reagent regions between a capture region (immobilized with another antibody capable of specifically binding to the analyte) and a site at which a test sample is absorbed in the test strip, wherein the first labeled antibody is obtained by labeling an antibody capable of specifically binding to an analyte, and the second labeled antibody is obtained by labeling a secondary antibody capable of specifically binding to the antibody. The analyte in the sample forms a complex with the first labeled antibody, and thereafter the second labeled antibody is further bound to the first labeled antibody, thereby forming a complex of [analyte-first labeled antibody-second labeled antibody]. The immunological complex is captured by an antibody immobilized on the capture region. Therefore, a signal amplified by the second labeled antibody is detected on the capture region.
However, at present, a sufficient detection sensitivity has not yet been obtained even by the signal-amplifying immunochromatography described above. Further, when a test sample is feces, urine, blood, or the like, there is necessitated additional procedures such as a step of suspending a sample in an appropriate buffer as a pretreatment and/or partial purification process comprising separating and removing heterogeneous substances in the test sample, so that it has a defect of lack of quickness.
Accordingly, an object of the present invention is to provide an immunological detection method relating to immunochromatography, the method capable of detecting an analyte more quickly and with high sensitivity, and a kit used therefor.
The present invention provides the following immunological detection methods and kits used therefor:
[1] an immunological detection method for detecting an analyte by using a water-absorbent substrate in which a capture region immobilized with a first immunochemical component capable of specifically binding to the analyte is positioned in a given region on a surface thereof, wherein the immunological detection method is characterized by the use of:
(1) a solution comprising a labeled immunochemical component (first labeled immunochemical component) comprising a second immunochemical component capable of specifically binding to the analyte and a labeling substance, wherein the labeling substance is bound to the second immunochemical component; and
(2) a solution comprising a labeled immunochemical component (second labeled immunochemical component) comprising a third immunochemical component capable of specifically binding to the second immunochemical component and a labeling substance, wherein the labeling substance is bound to the third immunochemical component;
[2] an immunological detection method comprising forming on a capture region an immunological complex in which an analyte in a test sample is sandwiched with a first immunochemical component capable of specifically binding to the analyte, and a labeled component (first labeled component), the first labeled component comprising a second immunochemical component capable of specifically binding to the analyte, a third immunochemical component incapable of binding to the analyte and a labeling substance, wherein the labeling substance is bound to the second and third immunochemical components, wherein the first immunochemical component is immobilized on the capture region positioned in a given region on a surface of a water-absorbent substrate; and determining a signal of the labeling substance on the capture region, characterized in that the method comprises forming an immunological complex in which a labeled component (second labeled component) is bound to the third immunochemical component present in a sandwiched immunological complex via a mediating substance, the second labeled component comprising a fourth immunochemical component capable of specifically binding to the third immunochemical component via the mediating substance, and a labeling substance, wherein the labeling substance is bound to the fourth immunochemical component, thereby amplifying the signal of the labeling substance;
[3] a sandwiched-type immunological detection method wherein at a capture region immobilized with a first immunochemical component capable of binding to an analyte, the analyte is sandwiched by the first immunochemical component and a labeled component comprising a second immunochemical component capable of binding to the analyte and a labeling substance, wherein the labeling substance is bound to the second immunochemical component, characterized in that the immunological detection method comprises forming a complex via binding between a biotin and an avidin, and detecting the analyte;
[4] a kit for immunological detection characterized in that the kit comprises a water-absorbent substrate in which a capture region immobilized with a first immunochemical component capable of specifically binding to an analyte is positioned in a given region on a surface thereof; a labeled immunochemical component (first labeled immunochemical component), the first labeled immunochemical component comprising a second immunochemical component capable of specifically binding to the analyte, and a labeling substance, wherein the labeling substance is bound to the second immunochemical component; and a labeled immunochemical component (second labeled immunochemical component), the second labeled immunochemical component comprising a third immunochemical component capable of specifically binding to the second immunochemical component, and a labeling substance, wherein the labeling substance is bound to the third immunochemical component;
[5] a kit for immunological detection comprising a water-absorbent substrate in which a capture region immobilized with a first immunochemical component capable of specifically binding to an analyte is positioned in a given region on a surface thereof; a labeled component (first labeled component), the first labeled immunochemical component comprising a second immunochemical component capable of specifically binding to the analyte, a third immunochemical component incapable of binding to the analyte, and a labeling substance, wherein the labeling substance is bound to the second and third immunochemical components; a labeled component (second labeled component), the second labeled component comprising a fourth immunochemical component capable of specifically binding to the third immunochemical component via a mediating substance, and a labeling substance, wherein the labeling substance is bound to the fourth immunochemical component; and a mediating substance for mediating binding of the third and fourth immunochemical components;
[6] a kit for immunological detection comprising an immunological test strip comprising a capture region immobilized with a first immunochemical component capable of binding to an analyte on a water-absorbent substrate; a labeled component comprising a labeling substance, a biotin and a second immunochemical component capable of binding to the analyte, wherein the labeling substance is bound to the biotin and the second immunochemical component; and an avidin;
[7] a kit for immunological detection comprising an immunological test strip comprising a capture region immobilized with a first immunochemical component capable of binding to an analyte on a water-absorbent substrate; a labeled component comprising a first labeling substance, and an avidin and a second immunochemical component capable of binding to the analyte, wherein the labeling substance is bound to the avidin and the second immunochemical component; and a conjugate comprising a biotin and a second labeling substance; and
[8] a kit for immunological detection comprising an immunological test strip comprising a capture region immobilized with a first immunochemical component capable of binding to an analyte on a water-absorbent substrate; a labeled component comprising a first labeling substance, and a biotin and a second immunochemical component capable of binding to the analyte, wherein the first labeling substance is bound to a biotin and the second immunochemical component; and a conjugate comprising an avidin and a second labeling substance.